| Objective:Autoimmune hepatitis (AIH) is a chronic inflammatory disease of the liver. Currently, corticosteroids, alone or in combination with azathioprine is the main treatment of the disease. However, not all the patients respond fully to the medication, and the long course of treatment is often associated with drug-related side effects. Thus, a novel effective method is required to control and cure this disease. Mesenchymal stem cells from bone marrow stroma are able to differentiate into multiple mesoderm-type cell lineages and play an important role in immune regulation. Studies have demonstrated that bone-marrow-derived mesenchymal stem cells (BMMSCs) have been successfully used in treatment of autoimmune diseases. The aim of this research is to evaluate the therapeutic effects of BMMSCs on ConA-induced hepatitis and to elucidate the possible mechanism involved.Materials and Methods:1.Cell preparation and culture.The BMMSCs were isolated from bone tissues of C57BL/6mice using whole bone marrow adherence method. Expression of surface antigens of the third generation of BMMSCs were detected by flow cytometer. Osteogenous potential was evaluated by alizarin red staining. Adipogenic potential was assessed by oil red0staining. The third generation of BMMSCs was labeled with CM-Dil in vitro and fluorescence labeling rate was recorded under the fluorescence inverted microscope.2.Establishment of AIH mice models. C57BL/6mice were divided into four groups randomly and were injected with10mg/kg-1,15mg/kg-1,20mg/kg-1ConA or phosphate buffered saline (PBS) for group A, group B and group C or control group respectively. Levels of serum liver enzymes, histopathology were investigated at8h after injection.3.Transplantation of CM-Dil labeled BMMSCs in AIH mice models. C57BL/6mice were divided into five groups randomly. At30min after ConA administration, mice were injected with1×105,5×105,1×106,1×107CM-Dil labeled BMMSCs or PBS for group Ⅰ, group Ⅱ, group Ⅲ, group Ⅳ or control group, respectively. Survival rates, levels of serum liver enzymes, titers of serum cytokines, histopathology and localization of BMMSCs were investigated at24h after injection.Results: 1.The attached cells were observed48hours after primary cells culture and the figures change into bunches and radial pattern at the third generation. BMMSCs were negative for CD34, but positive for CD29and CD90using flow cytometry. Following induction, alizarin red staining and oil red staining produced a positive reaction in cells. Twenty four hours after labeling with CM-Dil in vitro, cells gave out red light, and marked rates were over90%under the fluorescent microscope.2.After injection of different dosages of ConA, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and Knodell scores of the hepatitis were significantly higer in group A, group B and group C than those in normal group (P<0.05). Histological damage was detected only in the liver but not in the heart, lung or spleen.3.Four mice died in groupⅣ within24hours and mice in other groups were still alive. There was a significant reduction of ALT, AST, Knodell scores and cytokines in group II and group III compared to the control group at24h after injection. In contrast, there was no significant difference between group I and control group in the measurements above. Injected BMMSCs were located in the liver of ConA-injected mice, however, there was no labeled BMMSCs detected in the heart, lung or spleen.Conclusions:1.BMMSCs could separated, cultured and amplification in vitro. The procedure of CM-Dil labeling is simple, stable and convenience. It is an effective method for in vivo tracing of BMMSCs.2.Hepatitis models induced by ConA is simple and reliable. It can be used in the research of autoimmune hepatitis.3.The therapeutic efficacy of BMMSCs for ConA-induced hepatitis is defineted. It is possible that MMSCs migrate to the inflammatory sites and reduce the production of TNF-a, IFN-y and IL-4to alleviate liver injury induced by ConA. |
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